G-Protein Coupled Receptors (GPCRs) modulate the response of many drugs, hormones and neurotransmitters in biology. Many disorders and diseases are equally focussed around GPCR function, with therapeutics based on altering the responsivity of GPCR function by use of small ligands or peptides, acting as either agonists or antagonists.
More than 30% of all currently prescribed pharmaceutical drugs involve GPCR-mediated modulation, and more than 30% of all drug targets classes are aimed at understanding and modulating GPCR function.
GPCR Structure and Associated Binding Proteins
Currently, there are approximately 400 known GPCRs, characterised historically by nomenclature into Type A, (rhodopsin like), Type B (calcitonin) and Type C (metabotropic). With the advent of the sequencing of the human genome, sequence analysis and homology searching implied the presence of at least 150-200 GPCR-like proteins which currently possess no known endogenous ligands. These latter GPCRs are known as “orphan” receptors (‘oGPCR’, Howard et al. (2001) Trends in Pharm. Sci., 22, 132-140). In total, there are ˜400-500 endogenous ligands known to function via characterised GPCRs, including those for recently “de-orphanised” GPCRs.
GPCRs are characterised by a conserved seven-transmembrane spanning motif, which comprise of protein helices linked by both intracellular and extracellular loop domains. The extracellular domains of GPCRs contain ligand docking (binding) sequences, and the intracellular loop domains (2nd and 3rd loop) are important docking sites for GPCR-associated proteins (Moro et al. (2003) Chem. Commun., 24, 2949-2956).
In Nature, the ligand-binding event, which occurs at extracellular binding sites on the GPCR, is transduced, postulated to be via resultant protein conformational shifts, into the intracellular matrix. The transduction mechanism is signalled via an “early event” intracellular exchange of guanosine diphosphate (GDP) for GTP (guanosine triphosphate). GDP is present at the “resting”, or “ligand-unoccupied” state, and exchange for GTP occurs at the “active”, or “ligand-occupied” state. The binding of either GDP or GTP occurs at defined sites of an intracellular heterotrimeric complex, known as the G-proteins, which comprise 3 subunits, Gα, Gβ and Gγ. GTP and GDP bind to the Gα subunit of the heterotrimer. The G-protein complex resides on the intracellular face of membranes and is closely associated with residues within the intracellular loop domain of the GPCR. Coupling of most G protein-coupled receptors to heterotrimeric G proteins involves the third intracellular loop and the proximal region of the carboxyl-terminal tail of the GPCR.
Upon binding of GTP to the Gα subunit, there is a resultant perturbation of the G-protein complex, which subsequently induces downstream transduction via effector systems (e.g. such as phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolysis with subsequent changes in this instance, to intracellular Ca2+ (Ca2+i) levels). Ligand activation may also involve internalisation of the receptor (e.g. for downstream gene induction) or direct opening of ligand-gated ion channels.
At a later stage, regardless of the precise mechanism of ligand activation, there is a need for the response to be attenuated. To “decouple” or “downregulate” the response of ligand binding to the GPCR as well as attenuate subsequent downstream transduction events, the GTP-bound to the Gα subunit is hydrolysed by endogeneous enzymes (GTPases) back to guanosine diphosphate (GDP), leading to functional reassociation of the G-proteins and dissociation of the ligand with a return to the “resting” or “ligand-unoccupied” state.
GPCR-active ligands, and GPCR action in general, can also be characterised by the nature of the transduction event linked to Gα functionality. Gα functionality has been shown to be linked to primary sequences. It therefore follows that G protein classes can also be defined according to the primary sequences of their Gα subunits. This classification has lead to definition of 4 families: Gαs, Gαi/o, Gαq αnd Gα12/13.                Gαs (μ[cAMP] via adenylate cyclase activation)        Some GPCR-active ligands can be characterised by a downstream increase in intracellular concentration of adenylate cyclase, which leads to a subsequent increase in intracellular concentrations of the important second messenger, cyclic adenosine monophosphate (cAMP). Upon ligand binding, transduction in this case is via GDPGTP exchange at the Gαs subunit.        Gαi/o (∘[cAMP] via adenylate cyclase inhibition, or K+ channel modulation or phosphodiesterase (PDE) activation)—Upon ligand binding, transduction occurs via GDPGTP exchange at the Gαi subunit.        Gαq (protein coupled, μCa2+i via phospholipase C beta (PLCβ))        Transduction is via GDPGTP exchange at the Gαq subunit. Response is pertussis toxin sensitive. PLCβ catalyses release of diacylglycerol (DAG) and inositol 1,4,5-phosphate (IP3) from inositol 4,5-diphosphate (PIP2). IP3 increase is linked to Ca2+i release. It has also been found that heterologous expression of Gα16 (a member of Gαq), can allow coupling of a wide range of GPCRs to PLCβ activity and allow measurements of Ca2+i flux.        Gα12/13 (protein coupled, interacting with Cl− channels)        Transduction is via GDPGTP exchange at the Gα12/13 subunitMethods of Carrying Out GPCR Assays to Measure Ligand Potency        
There is a continuing desire within the pharmaceutical industry to exploit GPCRs and orphan GPCRs as drug targets. Many methods have been used to measure GPCR activity and in vitro assays form an important part of high throughput screening strategies in the search for new GPCR-active ligands. Complementary technologies involve cell-based assay formats in which for example, Ca2+i flux measurement can be made within intact cells by use of calcium-sensitive fluorescent indicators. In the latter case, the use of sensitive detection platforms have been aided by the creation of chimeric G proteins (such as Gαs-Gαq) or the heterologous expression of Gα16, to allow “forced coupling” of ligand response through PLCβ activation pathways, enabling a Ca2+i readout to be made (Milligan & Rees (1999) Trends in Pharm. Sci., 20, 118-124).
Traditional methods of carrying out GPCR assays involve use of radioactive ligands. These are employed in heterogeneous filter-based or homogeneous SPA-based (Scintillation Proximity Assay) assays. From these studies, the end user can obtain information on ligand potency by measurement of the radioactive counts on the filter (after separation of bound from free ligand) or directly on the SPA bead.
Use of Radioactive [35S]GtpγS
To exploit the binding of GTP to Gα as a high sensitivity in vitro assay interrogation point, researchers have developed GTPase-resistant (“non-hydrolysable”) analogues of GTP, with one of the most efficacious being radioactive [35S]GTPγS (Milligan (2003) Trends in Pharm. Sci., 24, 87-90; Ferrer et al. (2003) Assay & DDT, 1, 261-273). When a non-radioactive ligand now binds to cell membranes carrying a functional GPCR [35S]GTPγS is recruited to the G-protein Gα subunit. As [35S]GTPγS is essentially “non-hydrolysable”, the receptor/G-protein system is effectively “locked” in a ligand-occupied state. Now, radioactive filter counts or SPA counts of G-protein-bound [35S]GTPγS allows the user to obtain information on both the ligand binding potency as well as the ligand efficacy. Use of [35S]GTPγS in this manner means that, in essence, the user is carrying out an in vitro “functional assay”. The GTP-probe is effectively acting as a post-binding event reporter, at an early position in the transduction process.
The Need for Homogeneous Fluorescence Assays for GPCRs
Whilst inherently sensitive radioactive assays (heterogeneous and homogeneous format) have formed the bulk of generic in vitro screening assays for GPCRs, there has been a desire to move towards sensitive, non-radioactive, and in particular homogeneous assays (Kimble et al., (2003) Combin.Chem & High Thr. Screening, 6, 409-418). The latter assay formats are particularly amenable to miniaturisation and hence provide time and material cost savings. A robust signal which can be easily measured on a spectrophotometer, in particular an optical signal, would be of advantage. Fluorescence intensity measurements, and in particular Fluorescence Resonance Energy Transfer (FRET), would fulfil many desirable requirements for a suitable assay format.
FRET is a distancε-related process in which the electronic excited states of two dye molecules interact without emission of a photon (Forster, T., “Intermolecular Energy Transfer and Fluorescence”, Ann. Physik., Vol. 2, p. 55, (1948)). One result of this interaction is that excitation of a donor molecule enhances the fluorescence emission of an acceptor molecule. The fluorescence quantum yield of the donor is correspondingly diminished. For FRET to occur, suitably, the donor and acceptor dye molecules must be in close proximity (typically between 10-100 Å), since energy transfer efficiency decreases inversely as the 6th power of the distance (r) between the donor and acceptor molecules.
In FRET, molecules which act as FRET “donors” are allowed to interact with molecules which act as FRET “acceptors”. By donor, it is meant that the dye moiety is capable of absorbing energy from light and emits light at wavelength frequencies which are at least partly within the absorption spectrum of the acceptor. By acceptor, it is meant that the dye moiety is capable of absorbing energy at a wavelength emitted by a donor dye moiety.
If these donor and acceptors come into close contact within a critical distance, then FRET occurs and spectroscopic measurements taken at the emission wavelengths of the acceptor will give an indication of the magnitude of the FRET interaction. If the donor and acceptor fluors are allowed to come into close contact as a result of a biological interaction, then it follows that the magnitude of the FRET signal will be related to the magnitude of the biological interaction under scrutiny. Under suitable conditions, the closest molecular distances between the FRET partners can be calculated from the maximum FRET signal.
Fluorescent Analogues of GTP
There has always been a desire to develop non-radioactive (fluorescent) reporter analogues of [35S]GTPγS. Many have been described in the literature, but most suffer from high rates of hydrolysis and/or poor affinity for the G-proteins (McEwen et al. (2001) Anal. Biochem., 291, 107-117); Korlach et al., (2004) Proc. Natl.Acad. Sci., 101, 2800-2805). There is, therefore, a need within the pharmaceutical industries for a hydrolytically stable fluorescent reporter analogue which has a high degree of affinity for the G-proteins. Such a reporter molecule is described herein and is the subject of the Applicant's (Amersham Biosciences UK Limited) co-pending patent application entitled ‘Fluorescent Nucleotide Analogues’ (WO 05/003685 claiming priority to applications GB 0421691.7 and GB 0500504.6).
Prior Art—Examples of “Intermolecular” GPCR Fret Assays
Both in vitro and cell-based GPCR FRET assays have been cited in the literature. The FRET interaction in these instances is between two interacting “partner” biological species (for example, proteins) with the “donor” and “acceptor” fluorescent molecules bound to their respective but separate, species. When the two biological partners interact, FRET can occur under controlled conditions.
As referred to herein, the term “intermolecular interactions” are described as those occurring between separate G-protein subunits, Gα, Gβ and Gγ.
Leaney et al., (J. Biol. Chem. (2002) 277, 28803-28809) describes the potential use of cyan fluorescently tagged Gα-protein subunits in FRET assays for investigating protein-protein interactions. Similarly, WO 03/008435 postulates on the use of Gα-green fluorescent protein (GFP) constructs in FRET assays for screening for GPCR drug targets. A method for detecting ligand binding using a FRET assay based upon the interaction of a blue fluorescent protein-Gα construct with a yellow fluorescent protein-Gα construct is reported in WO 02/077200 for identifying proteins involved in olfaction.
Bunemann et al., (Proc. Natl.Acad. Sci. (2003) 26, 16077-16082) describe use of cloned fluorescent protein tagged G-proteins which were viably reconstituted into cultured host human embryonic kidney (HEK) cells. G-proteins were tagged with either cyan fluorescent protein (CFP) or yellow fluorescent protein (eYFP), namely, Gαi-eYFP, Gβ1-CFP and/or Gγ2-CFP. FRET signals were observed that were ligand (agonist) dependent, and which were postulated to be as a result of G-protein conformational shifts in response to specific ligand binding, allowing a measure of both ligand binding potency as well as changes in intermolecular distances, as the ligand “on-off” cycle progresses.
Potential limitations pertaining to this latter “intermolecular” strategy is the requirement for two (or more) species to interact at appropriate times, orientations and concentrations. Significant alteration of the endogenous G-proteins by attachment of large fluorescent proteins may well lead to perturbation of the binding events under investigation. Alternatively, random chemical labelling with smaller, low molecular weight (MW) fluorescent tags can be carried out, but this may also lead to perturbation of natural biological function due to for example, unwanted chemical modifications at key binding sites and subsequent attenuation of binding affinity. There is also a real possibility of an increase in non-specific binding interactions when more than one species is required for an interaction. Also, the creation of two or more binding partners each labelled with potentially large fluorescent proteins may for example, lead to severe steric interactions leading to an attenuated or anomalous FRET response.
In addition, two biological species (Gα and Gβ or Gγ in the example cited) have to be labelled with FRET partners, and if the labelling is intrinsic, then suitable cloning vectors have to be constructed leading to the generation of two or more recombinant proteins. To counter this situation by use of extrinsic labelling strategies, each binding partner may have be individually and site-specifically chemically labelled, which may be cumbersome, due to the requirements of high chemo- and regio-selectivity control.
Blaesius et al., (Presentation Number 135 in Session on ‘Assay Development and Validation Strategies’, The Society for Biomolecular Screening-7th Annual Conference, 12 Sep. 2001) and WO 04/035614 describe another example of an “intermolecular” FRET assay for GPCRs (FIG. 1), using engineered peptide affinity probes. The authors describe the use of novel biotinylated peptide affinity probes which differentiate the GTP-bound state from the GDP-bound state of Gαi or Gαs. By using a carboxy-terminal histidine-tagged (his6) reconstituted GPCR, they were able to show, upon addition of suitable GPCR ligands, a detectable FRET signal between streptavidin-europium (bound to the biotin affinity peptide) and an allophycocyanin (APC)-labelled anti-histidine antibody.
A limitation pertaining to this method is the fact that the peptide affinity probe is not covalently bound to the target Gα subunit, and therefore the relative affinities of a set of peptide probes need to be carefully evaluated for each set of Gα species. This is an important issue, as there are multiple types of Gαs, Gαi/o and Gαq that have been identified. Screening for sets of peptide probes of sufficient affinity is a laborious process, involving many cycles of compound generation and screening, such as by phage display panning, as well as optimisation by evolved library strategies. Indeed, even after suitable screening, the affinity of the peptide probe may either be quite low or be too highly cross-reactive between Gα species, so precluding use in an assay.
In addition, both the nature and position of binding of the probe is unknown, which is not an ideal situation when trying to optimise probe design. Another disadvantage of having a non-covalent binding probe is the risk of facile perturbation of this probe-target interaction by other factors, such as putative drugs or buffer/detergent conditions.
“Intramolecular” GPCR FRET Assays
Work by Frang et al., (‘Homogeneous GTP binding assay for GPCRs based on TR-FRET’, poster, SBS 9th Annual Conference, Portland, Oreg.), uses identical peptide affinity probe technology invented by Blaesius et al. (Karo Bio, USA Inc.) described above. Frang describes use of a biotinylated peptide sourced from Karo Bio, which is a peptide affinity probe that recognises the GTP-bound state of Gαi. FRET occurs as a result of interaction between streptavidin-europium donor label (bound to the biotin affinity probe) and a fluorescent GTP analogue (Alexa647-GTP), which acts as a FRET acceptor.
Although the FRET response is configured around a single biological molecular entity (Gαi in this case), and can therefore be referred to as “intramolecular”, the arguments cited earlier against employing the non-covalent binding of a biotinylated peptide affinity probe are still pertinent. Indeed, the arguments can be applied to any non-covalent binding approaches using any other type of probe, such as an antibody or aptamer.
The present invention seeks to address the above problems which exist in the prior art and to provide methods for detecting binding of a test compound (or ligand) to a GPCR and methods of identifying agents which modulate the binding of test compounds (or ligands) to GPCRs.